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. 2015 Oct 27;1:15030. doi: 10.1038/celldisc.2015.30

Figure 7.

Figure 7

(a) A375 melanoma cells were transiently transfected with 0.5 μg of NF-κB promoter and 0.05 μg of pCMVβGal to control the variability in transfection efficiency. Cells were treated for 48 h with vemurafenib 2 μm (Vem), BMS-345541 (1 μm) and TNFα (10 nm) or combinations as indicated. Luciferase activity was normalized to β-galactosidase activity and the results were expressed as fold stimulation of the basal luciferase activity from unstimulated cells. Data are means±s.d. of five experiments performed in triplicate. (**P⩽0.01, ***P⩽0.001) versus control. (b and c) A375 melanoma cells transfected with 50 nm of either siRNA control (SiCt) or siRNA IKK (SiIKK) were exposed or not to vemurafenib (2 μm) or TNFα (10 nm) for 24 h. Proteins were extracted and CD271, IKK, IKβα and ERK expression were evaluated by western blot. ERK was used as a loading control (b). In parallel, the percentage of CD271 was determined by flow cytometry (c). (d) A375 melanoma cells were treated with vemurafenib at indicated concentration for 48 h and TNFα secretion in medium was detected by enzyme-linked immunosorbent assay. TNFα was used as a positive control. (e and f) A375 melanoma cells transfected with 50 nm of either siRNA control (SiCt) or siRNA IKK (SiIKK) were exposed or not to vemurafenib (2 μm) or TNFα (10 nm) for 48 h in the presence or the absence of 10 ng of TNFα-blocking antibody. Proteins were extracted and CD271, IKK, IKβα and ERK expression were evaluated by western blot (e) and the percentage of CD271 was determined by flow cytometry (f). (g and h) Skmel28-sensitive (Skmel28_S) and -resistant (Skmel28_R) melanoma cells transfected with 50 nm of either siRNA control (SiCt) or siRNA IKK (SiIKK) were exposed or not to vemurafenib (2 μm) or TNFα (10 nm) for 24 h. Proteins were extracted and CD271, IKK, IKβα and ERK expression were evaluated by western blot (g) and cell viability was determined using trypan blue dye exclusion method (h). The data showed the mean±s.d. of three independent experiments versus control (*P⩽0.05; **P⩽0.01; ***P⩽0.001).